A promoter is a DNA sequence, which influences or determines the expression location and expression quantity of a gene and makes available points for the bonding of RNA polymerase. The position of a promoter is fixed relative to the transcription starting point in the genome of an organism. RNA polymerase is an enzyme, which can connect to the promoter and puts into effect the transcription of a gene, which is under the control of said promoter. This leads to messenger RNA (mRNA), which is in turn used for protein synthesis.
Promoters have been investigated in various organisms. For certain species it was possible to find conserved DNA regions (so-called consensus sequences) within promoters, which are associated with different genes. It is assumed that these regions are bound into the part played by the promoter in the transcription process. The initiation of the transcription process in plants incorporates an interaction of the promoter with the RNA polymerase II. Concensus sequences were found in plant promoters above the 5′ end of the transcription starting point. One of these sequences is approximately 7 base pairs long and is approximately 20 to 30 base pairs above the transcription starting point. This sequence is known as a so-called TATA box and it is assumed that it plays a part in RNA polymerase bonding. Another sequence with a length of approximately 9 base pairs is located approximately 70 to 90 base pairs above the transcription starting point. This sequence is called the CAAT box and it is assumed that it plays a part in regulating the transcription level. Other regions above the transcription starting point have been identified which influence the frequency of transcription initiation in eukaryons. These DNA regions, known as enhancers, influence the activity of promoters in their vicinity. However, by definition, these sequences are not promoters, because their position does not have to be fixed.
In order to be able to express a foreign gene in an organism, e.g. a plant, the coding sequence of this gene must be placed under the control of a promoter and introduced into the plant. For inserting the gene to be expressed in the plant genome, the foreign DNA is usually brought into the Ti-plasmide of agrobacterium tumefaciens and the latter is then used for transforming the plants. A second, frequently used method is the direct transformation of DNA, e.g. with the aid of the “particle gun”. Up to now, in most cases for this purpose use has been made of promoters isolated from bacteria or promoters of plant viruses, which lead to the expression of the foreign gene in the plants. For certain applications these promoters suffer from the disadvantage that they are of a different species and are consequently not subject to the control mechanisms within the plants.
When using a plant promoter it is possible to express a foreign gene, which is consequently also subject to the plant control mechanisms. By testing the expression of the gene in front of which the promoter was originally located, precise information can be obtained regarding the expression intensity, the time at which the gene is expressed and the expression location and these can be largely transferred to the expression of a foreign gene, placed under the control of this promoter. Another advantage is that when using a precisely characterized, plant promoter, planned interventions and research on the development of certain plant parts are possible.
Problem and Solution
The problem of the present invention is to make available a promoter, which is suitable for controlling the expression of nucleic acids in plants or plant cells. A partial aspect of the problem is to make available promoters having a high expression and simultaneously tissue specificity. In another aspect, the problem of the invention is to provide a process for the production of male, sterile plants.
One aspect of the problem of the invention is solved by a nucleic acid sequence coding for a promoter, which is both tapetum-specific and pollen-specific.
In a second aspect the problem is solved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence covering a range of at least approximately 900 nucleotides upstream of the TATA box of the sequence represented in SEQ ID No. 1.
According to an embodiment, the nucleic acid sequence covers a range of at least approximately 1,000 nucleotides upstream of the TATA box of the sequence represented in SEQ ID No. 1.
According to another embodiment, the nucleic acid sequence covers a range of at least approximately 1,500 nucleotides upstream of the TATA box of the sequence represented in SEQ ID No. 1.
In yet another embodiment, the nucleic acid sequence covers the sequence represented in SEQ ID No. 1.
According to another aspect this problem is also solved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence covering the sequence represented in SEQ ID No. 2.
In a fourth aspect the problem is solved by a nucleic acid sequence coding for a promoter, the nucleic acid sequence covering the sequence represented in SEQ ID No. 3.
In a fifth aspect the problem is solved by an expression system covering at least one of the nucleic acids according to the invention.
According to an embodiment, the expression system comprises at least one terminator and/or a linker.
In a sixth aspect the problem is solved by a nucleic acid construct comprising a nucleic acid sequence according to the invention and at least part of an expressible nucleic acid sequence.
According to an embodiment, the part of the expressible nucleic acid sequence or the complete, expressible sequence is linked with one of the nucleic acid sequences according to the invention in the sense direction.
In a preferred embodiment, the expressible nucleic acid codes for an invertase.
According to another preferred embodiment, the part of the nucleic acid sequence of an invertase or the complete sequence of an invertase is connected with one of the nucleic acid sequences according to the invention in the antisense direction.
According to an embodiment the invertase is of the type present in a structure selected from the group comprising anthers, tapetum, pollen precursor cells and pollen.
In another embodiment the invertase comes from the organism into which or into whose cells the nucleic acid construct is to be introduced and in particular from the plant group to which the species to be introduced into the nucleic acid construct belongs.
In yet another embodiment the organism is selected from the group comprising food plants, ornamental plants and medicinal plants.
In a seventh aspect the problem is solved by a vector comprising one of the nucleic acid sequences according to the invention and/or an expression system 9 according to the invention and/or a nucleic acid construct according to the invention.
In an eighth aspect the problem is solved by a cell, particularly a plant cell, comprising a nucleic acid according to the invention and/or an expression system according to the invention and/or a nucleic acid construct according to the invention and/or a vector according to the invention.
According to an embodiment the cell comprises a nucleic acid sequence according to the invention, which is a promoter, and a nucleic acid coding for an inhibitor of an invertase, the promoter controlling the expression of the inhibitor.
According to another embodiment the cell is selected from the group comprising pollen cells, pollen precursor cells and tapetum cells.
In a particularly preferred embodiment the cell is an arrested pollen cell.
In a ninth aspect the problem is solved by a plant incorporating a cell according to the invention.
According to an embodiment the plant is selected from the group comprising food plants, ornamental plants and medicinal plants, preferably chosen from the group comprising rice, maize, potatoes, tomatoes and rape.
In a further embodiment the plant is a male, sterile plant and has at least one further modification of its genotype, particularly a genetically engineering-caused change.
In a tenth aspect the problem is solved by a seed obtainable from a plant according to the invention.
In an eleventh aspect the problem is solved by a hybrid seed obtainable by hybridizing a male, sterile plant according to the invention with another male, fertile plant and the hybrid seed is obtained from the resulting filial generation.
In a twelfth aspect the problem is solved by a process for the production of male, sterile plants, a nucleic acid construct according to the invention being introduced into a cell, particularly into a plant cell and from said cell a plant is produced.
In an embodiment the plant is selected from the group comprising food, ornamental and medicinal plants, preferably selected from the group comprising rice, maize, potatoes, tomatoes and rape.
In a thirteenth aspect the problem is solved by the use of a nucleic acid construct according to the invention for producing male, sterile plants.
In a fourteenth aspect the problem is solved by the use of a nucleic acid sequence according to the invention for the expression of a nucleic acid sequence.
In a fifteenth aspect the problem is solved by a restorer plant, incorporating in a cell, preferably in most of its cells, a nucleic acid according to the invention as a promoter and a nucleic acid coding for a further invertase, which is controlled by said promoter, the further invertase being different from the cell's own invertase.
In a sixteenth aspect the problem is solved by a restorer plant, which can preferably be of the above-described type comprising in a cell and preferably in most of its cells, a nucleic acid according to the invention as a promoter and a nucleic acid coding for a saccharose transport system and which is controlled by said promoter.
According to an embodiment in a cell and preferably in most of its cells, it also incorporates a nucleic acid according to the invention as a promoter and a nucleic acid coding for saccharose synthase and/or and/or cytoplasmically expressed invertase, whose expression is controlled by the promoter.
In a seventeenth aspect the problem is solved by a plant, which is characterized in that in at least one cell and preferably in most of its cells it incorporates a nucleic acid construct according to the invention and the cell or cells also comprise a nucleic acid sequence according to the invention as a promoter and a nucleic acid coding for a further invertase and which is controlled by said promoter, the further invertase differing from the cell's own invertase.
In an eighteenth aspect the problem is solved by a plant, which is characterized in that in at least one cell and preferably in most of its cells, it incorporates a nucleic acid construct according to the invention and the cell or cells also comprise a nucleic acid sequence according to the invention as a promoter and a nucleic acid coding for a saccharose transport system and which is controlled by said promoter.
In a preferred embodiment the plant also comprises the features of the plant according to the seventeenth aspect of the present invention.
According to a further embodiment the plant comprises in at least one cell and preferably in most of its cells a nucleic acid construct according to the invention and the cell or cells also comprise a nucleic acid sequence according to the invention as a promoter and a nucleic acid coding for saccharose synthase and/or cytoplasmically expressed invertase, whose expression is controlled by the promoter.
In yet another embodiment the further invertase differing from the cell's own invertase is selected from the group of invertases incorporating invertase(s) of Saccharomyces cerevisiae and invertase(s) of Zymomonas mobilis. 
According to another embodiment the saccharose synthase is of a heterologous or homologous origin.
In a further embodiment the cytoplasmically expressed invertase is of a homologous or heterologous origin.
In a particularly preferred embodiment the cytoplasmically expressed invertase is of heterologous origin and is preferably selected from the group of invertases including invertase(s) of Saccharomyces cerevisiae and invertase(s) of Zymomonas mobilis. 
In a nineteenth aspect the problem is solved by a seed obtainable from a plant according to the invention.
In a twentieth aspect the problem is solved by the use of seed according to the invention for the in vitro embryogenesis of haploid or diploid or double diploid plants.
In a twenty first aspect the problem is solved by a fruit, particularly a seedless fruit obtainable from one of the plants according to the invention.
In a twenty second aspect the problem is solved by a fruit obtainable from one of the plants according to the invention and in particular from a restorer plant according to the invention and its hybridization products according to the invention.
In a twenty third aspect the problem is solved by a process for cloning promoters, which are functionally homologous to one of the promoters according to one of the preceding claims, the process being characterized by the following steps:                a) cloning of anther-specific invertase cDNA by RT-PCR on mRNA from anthers, particularly using oligonucleotides OIN3 and OIN4,        b) cloning the corresponding promoters.        
The present invention is based on the surprising finding that promoters exist, which are suitable for the expression of nucleic acids in plant cells and have a double tissue specificity. The nucleic acids disclosed, which code for a promoter and which are referred to hereinafter as promoters for short, have an at least double specificity. They lead to the expression of the nucleic acid under their control in the tapetum and pollen. In addition, the promoters according to the invention are particularly strong and have a characteristic path over anther evolution subdivided into 12 phases and which makes it possible when using promoters according to the invention to obtain a time-defined, specific expression pattern. As a result of this both spatial specificity, i.e. tissue specificity, and time specificity such vectors offer a major advantage compared with promoters inducible by an exogenous stimulus, such as temperature or the presence of certain compounds. If such promoters are contained in the genome of a plant, there is a time and space-specific expression of the nucleic acid(s) under the control of said promoter.
The nucleic acid under the control of the promoter according to the invention can be any nucleic acid form. Correspondingly they can be coding nucleic acids or structural or functional nucleic acids.
The term coding nucleic acid is understood to mean more particularly a nucleic acid coding for a peptide or protein. The peptide/protein can e.g. be a structural protein or a peptide/protein having enzymatic activity.
A structural nucleic acid is more particularly understood to mean a nucleic acid leading to the formation of complexes, particularly with other molecules. It can inter alia be a rRNA and in particular an antisense nucleic acid.
A functional nucleic acid is more particularly understood to mean a nucleic acid, which exerts a specific action on a system, particularly a biological system. Such a specific action can e.g. be the aiding or inhibiting of translation or transcription. An example of a functional nucleic acid is an antisense nucleic acid.
It is clear to the expert that the above definitions relate to different aspects of nucleic acids and consequently do not represent exclusive definitions. It is in fact possible for the same nucleic acid to be covered by two or more of these definitions.
The promoters according to the invention permit the space and time-determined expression of nucleic acids, particularly of genes in plant cells and plants. These can be homologous or heterologous nucleic acids or genes. The homologous genes are those obtained from the genetic background of the plant containing one of the promoters according to the invention. Thus, the genes or nucleic acid sequences already present in the cell are either additionally or alternatively placed under the control of the promoters according to the invention. The heterologous genes or nucleic acids are those not coming from or present in the genetic background of the plant containing one of the promoters according to the invention.
The invention is also based on the surprising finding that it is possible to produce male, sterile plants, particularly when using one of the promoters according to the invention. For this purpose a nucleic acid coding for at least part of an invertase is placed under the control of one of the promoters according to the invention. The invertase is preferably of the type present in pollen and/or tapetum and which can come from the given plant species. Particular preference is given to the nucleic acid sequence according to SEQ ID No. 15 or part thereof. The invertase coded by SEQ ID No. 15 is of the type isolated from tobacco pollen. Part of the nucleic acid coding this invertase is brought under the control of the promoter according to the invention, so that the expression product of the nucleic acid coding the invertase acts as antisense nucleic acid and subsequently suppresses the expression of the invertase present in the pollen and tapetum. The antisense nucleic acid is produced in that the nucleic acid coding for the invertase or a part thereof is functionally coupled in the antisense direction to the promoter, optionally separated by an additional nucleic acid sequence, e.g. in the form of a linker. This is implemented in that the non-coding or antisense strand is read by the promoter and consequently the nucleic acid coding the invertase is incorporated in inverted form.
As will be shown hereinafter, under the influence of such a construct sterile pollen or sterile, male plants are formed. Without wishing to be bound by this, it would appear that as a result of the antisense nucleic acid the expression of the invertase in tapetum and in pollen is suppressed in that the antisense nucleic acid interacts with the sense nucleic acid, which is read by the gene of the invertase present in said tissues and consequently a translation no longer takes place. As a result the invertase titre in the tissues drops, so that firstly there is an energetic deficiency, particularly in the pollen and also the ratio of disaccharide, particularly saccharose, to one or both the monomers thereof changes. This leads to the observed infertility of the pollen and consequently the male infertility of plants carrying such pollen.
With respect to this mechanism it is significant that as a result of the tissue-specific and time expression pattern of the vectors according to the invention, the antisense nucleic acid specifically occurs if the invertase in said tissues is particularly active and must be suppressed in order to bring about the above-described, energetic deficiency and/or the shift of the disaccharide to monosaccharide ratio. As a result of the strength of the promoters according to the invention the antisense nucleic acid is expressed to such a significant extent that there is an effective suppression of the intrinsic invertase activity in pollen and tapetum. As a result of this sterile pollen and male, sterile plant formation mechanism, the pollen is arrested in a clearly defined stage of its development. This specific stage is referred to as the mononuclear microspore stage, which otherwise fertile pollen would pass through within the scope of normal development.
This mechanism for producing male sterility in plants not only occurs in tobacco or tomatoes. The promoter can in fact be used in any plant or plant species. The same applies in principle for nucleic acid coding for an invertase or part thereof functionally linked in antisense orientation with one of the promoters according to the invention. As, due to the mechanism, there is an interaction between the invertase intrinsically contained in the pollen (or the extracellular invertase produced by the same) or the nucleic acid coding it, particularly mRNA, it is advantageous in the corresponding constructs according to the invention to so select the invertase sequence used in the construct that it is identical with the sequence of the intrinsic invertase or has a degree of homology therewith allowing an interaction of the sense and antisense nucleic acid.
Another mechanism for producing male, sterile plants is co-suppression. The term co-suppression is understood to mean the effect that in the case of the overexpression of a gene already present in a plant, it does not lead to an increased formation of the peptide/protein coded by the gene and instead leads to a reduced formation. The action consequently corresponds to the antisense construct described herein comprising one of the promoters according to the invention and a nucleic acid, coding for an invertase and functionally coupled thereto in antisense orientation. Co-suppression evolves its action on the transcription plane. As a result the considerations made in connection with antisense construction concerning homologies of nucleic acid under one of the promoters according to the invention also apply here. Thus, this mechanism also represents a use possibility for the nucleic acid constructs disclosed herein, where a nucleic acid is bound in the sense orientation to one of the promoters according to the invention.
The usability of mechanisms according to the invention for the production of male sterility or for producing male, sterile plants is not restricted to specific plant families, types or species and it is instead a universally usable mechanism. Correspondingly plants are here understood to mean in general and in particular food, ornamental and medicinal plants. In the sense of the present invention, plants relate both to monocotyledons and dicotyledons, which present plant groups in the sense of the present invention. The use of invertases can extend both to invertases of or in both monocotyledons and dicotyledons. Despite the homology of invertases of monocotyledons and dicotyledons, there are distinct differences, which can be significant for construction or use. Another preferred group of plants, where different aspects of the invention can be applied or used, are rice maize, tomatoes, potatoes, rape, soya and sugar beet.